CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is a chloride channel that plays a critical role in secretion and absorption of water and electrolytes across epithelia. Since CFTR channels are also expressed in cardiac myocytes and are found to shorten action potential duration and induce repetitive activity, they are implicated to be arrythmogenic. One unique feature of the CFTR channel as an ion channel is that the free energy of ATP hydrolysis is harvested to drive the conformational changes that open and close the channel. Studies using mutant CFTR and various ATP or phosphate analogs have suggested a model that ATP hydrolysis at two nucleotide binding sites is tightly coupled to the opening and closing of the channel pore. Our understanding of the molecular basis of the coupling mechanism, however, remains primitive. Unresolved questions include: What is the stoichiometry of ATP binding/hydrolysis to gating transitions? How are the biochemical states in ATP hydrolysis cycles translated to the open and closed states in the gating transitions? Which part of the protein forms the aqueous pore? What is the relationship between the gate and the pore? These are fundamental questions that interest a broad spectrum of physiologists. A combinational approach is being adopted to tackle the molecular physiology of CFTR chloride channels. Different configurations of the patch-clamp techniques will be used to record CFTR channel activity so that both the cytoplasmic and the extracellular side of the channel are accessible to channel blockers, modifiers, or channel openers. Mutations in critical regions of the protein will be made to study the functional consequences of single amino acid substitutions on gating and permeation/blocking. State-dependent chemical modifications of engineered cysteines allow us to explore the dynamic protein conformational changes during gating transitions.
The specific aims of the project are:
Aim 1. To understand how ATP hydrolysis is coupled to the opening and closing transitions of CFTR.
Aim 2. To probe the CFTR pore with permeant and impermeant anions.
Aim 3, To study the structure-function relationship between the gate and the pore of CFTR. A clear understanding of the molecular mechanisms of CFTR function will aid in the design of pharmacological agents for therapeutic intervention in cystic fibrosis, secretory diarrhea and cardiac arrythmia.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL053445-08
Application #
6698009
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (01))
Program Officer
Wang, Lan-Hsiang
Project Start
1996-04-19
Project End
2005-11-30
Budget Start
2003-12-01
Budget End
2005-11-30
Support Year
8
Fiscal Year
2004
Total Cost
$253,750
Indirect Cost
Name
University of Missouri-Columbia
Department
Type
Organized Research Units
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Hwang, Tzyh-Chang; Kirk, Kevin L (2013) The CFTR ion channel: gating, regulation, and anion permeation. Cold Spring Harb Perspect Med 3:a009498
Sohma, Yoshiro; Yu, Ying-Chun; Hwang, Tzyh-Chang (2013) Curcumin and genistein: the combined effects on disease-associated CFTR mutants and their clinical implications. Curr Pharm Des 19:3521-8
Jih, Kang-Yang; Hwang, Tzyh-Chang (2012) Nonequilibrium gating of CFTR on an equilibrium theme. Physiology (Bethesda) 27:351-61
Jih, Kang-Yang; Sohma, Yoshiro; Li, Min et al. (2012) Identification of a novel post-hydrolytic state in CFTR gating. J Gen Physiol 139:359-70
Bai, Yonghong; Li, Min; Hwang, Tzyh-Chang (2011) Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7). J Gen Physiol 138:495-507
Cai, Zhiwei; Sohma, Yoshiro; Bompadre, Silvia G et al. (2011) Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants. Methods Mol Biol 741:419-41
Tsai, Ming-Feng; Jih, Kang-Yang; Shimizu, Hiroyasu et al. (2010) Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels. J Biol Chem 285:37663-71
Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang (2010) Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel. J Gen Physiol 135:399-414
Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi et al. (2010) A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR. J Physiol Sci 60:353-62
Huang, Sheng-You; Bolser, Diana; Liu, Hao-Yang et al. (2009) Molecular modeling of the heterodimer of human CFTR's nucleotide-binding domains using a protein-protein docking approach. J Mol Graph Model 27:822-8

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